This invention relates to heterobifunctional poly(alkylene oxides) having degradable linkages and to conjugates derived therefrom.
In its most common form, the poly(alkylene oxide) poly(ethylene glycol) (PEG) is a linear polymer terminated at each end with hydroxyl groups:
HOxe2x80x94CH2CH2Oxe2x80x94(CH2CH2O)nxe2x80x94CH2CH2xe2x80x94OH
This polymer can be represented in a brief form as HOxe2x80x94PEGxe2x80x94OH where it is understood that xe2x80x94PEGxe2x80x94 represents the following structural unit:
xe2x80x94CH2CH2Oxe2x80x94(CH2CH2O)nxe2x80x94CH2CH2xe2x80x94
where n typically ranges from approximately 10 to 2000.
PEG is of great utility in a variety of biotechnical and pharmaceutical applications, particularly for drug delivery and modification of drug surfaces to promote nonfouling characteristics.
PEG is not toxic, does not tend to promote an immune response, and is soluble in water and in many organic solvents. The PEG polymer can be covalently attached to insoluble molecules to make the resulting PEG-molecule conjugate soluble. For example, Greenwald, Pendri and Bolikal in J. Org. Chem., 60, 331-336 (1995) recite that the water-insoluble drug taxol, when coupled to PEG, becomes water soluble. Davis et al. in U.S. Pat. No. 4,179,337 recite that proteins coupled to PEG have an enhanced blood circulation lifetime because of a reduced rate of kidney clearance and reduced immunogenicity. The lack of toxicity of the polymer and its rate of clearance from the body are important considerations in pharmaceutical applications. Pharmaceutical applications and many leading references are described in the book by Harris (J. M. Harris, Ed., xe2x80x9cBiomedical and Biotechnical Applications of Polyethylene Glycol Chemistry, Plenum, N.Y., 1992).
PEG is commonly used as methoxy-PEGxe2x80x94OH, or mPEG in brief, in which one terminus is the relatively inert methoxy group, while the other terminus is a hydroxyl group that is subject to ready chemical modification
CH3Oxe2x80x94(CH2CH2O)nxe2x80x94CH2CH2xe2x80x94OH mPEG
PEG is also commonly used in branched forms that can be prepared by addition of ethylene oxide to various polyols, including glycerol, pentaerythritol and sorbitol. For example, the four-armed branched PEG prepared from pentaerythritol is shown below:
C(CH2OH)n+n C2H4Oxe2x86x92C[CH2Oxe2x80x94(CH2CH2O)nxe2x80x94CH2CH2xe2x80x94OH]4
The branched PEGs can be represented in a general form as R(xe2x80x94PEGxe2x80x94OH)n in which R represents the central core molecule, which can include, e.g., glycerol or pentaerythritol, and n represents the number of arms.
Often it is necessary to use an xe2x80x9cactivated derivativexe2x80x9d of PEG to couple PEG to a molecule. The hydroxyl group located at the PEG terminus, or other group subject to ready chemical modification, is activated by modifying or replacing the group with a functional group suitable for reacting with a group on another molecule, including, e.g., proteins, surfaces, enzymes, and others. For example, the succinimidyl xe2x80x9cactive esterxe2x80x9d of carboxymethylated PEG forms covalent bonds with amino groups on proteins as described by K. Iwasaki and Y. Iwashita in U.S. Pat. No. 4,670,417. The synthesis described in U.S. Pat. No. 4,670,417 is illustrated below with the active ester reacting with amino groups of a protein in which the succinimidyl group is represented as NHS and the protein is represented as PROxe2x80x94NH2:
PEGxe2x80x94Oxe2x80x94CH2xe2x80x94CO2xe2x80x94NHS+PROxe2x80x94NH2xe2x86x92PEGxe2x80x94Oxe2x80x94CH2xe2x80x94CO2xe2x80x94NHxe2x80x94PRO
Succinimidyl xe2x80x9cactive estersxe2x80x9d, such as PEGxe2x80x94OCH2xe2x80x94CO2xe2x80x94NHS, are commonly used forms of activated carboxylic acid PEGs, and they are prepared by reacting carboxylic acid PEGs with N-hydroxysuccinimide.
PEG hydrogels, which are water-swollen gels, have been used for wound covering and drug delivery. PEG hydrogels are prepared by incorporating the soluble, hydrophilic polymer into a chemically crosslinked network or matrix so that addition of water produces an insoluble, swollen gel. Substances useful as drugs typically are not covalently attached to the PEG hydrogel for in vivo delivery. Instead, the substances are trapped within the crosslinked matrix and pass through the interstices in the matrix. The insoluble matrix can remain in the body indefinitely, and control of the release of the drug typically can be somewhat imprecise.
One approach to preparation of these hydrogels is described by Embrey and Grant in U.S. Pat. No. 4,894,238. The ends of the linear polymer are connected by various strong, nondegradable chemical linkages. For example, linear PEG is incorporated into a crosslinked network by reacting with a triol and a diisocyanate to form hydrolytically stable urethane linkages that are nondegradable in water.
A related approach for preparation of PEG hydrogels has been described by Gayet and Fortier in J. Controlled Release, 38, 177-184 (1996) in which linear PEG was activated as the p-nitrophenylcarbonate and crosslinked by reaction with a protein, bovine serum albumin. The linkages formed are hydrolytically stable urethane groups and the hydrogels are nondegradable in water.
In another approach, described by N. S. Chu in U.S. Pat. No. 3,963,805, nondegradable PEG networks have been prepared by random entanglement of PEG chains with other polymers formed by use of free radical initiators mixed with multifunctional monomers. P. A. King described nondegradable PEG hydrogels in U.S. Pat. No. 3,149,006 that have been prepared by radiation-induced crosslinking of high molecular weight PEG.
Nagaoka et al. described in U.S. Pat. No. 4,424,311 preparing PEG hydrogels by copolymerization of PEG methacrylate with other comonomers such as methyl methacrylate. Vinyl polymerization produces a polyethylene backbone with PEG attached. The methyl methacrylate comonomer is added to give the gel additional physical strength.
Sawhney et al. described, in Macromolecules, 26, 581 (1993) and U.S. Pat. No. 5,626,863, the preparation of block copolymers of polyglycolide or polylactide and PEG that are terminated with acrylate groups:
CH2xe2x95x90CHxe2x80x94COxe2x80x94(Oxe2x80x94CHRxe2x80x94CO)nxe2x80x94Oxe2x80x94PEGxe2x80x94Oxe2x80x94(COxe2x80x94CHRxe2x80x94O)nxe2x80x94OCxe2x80x94CHxe2x95x90CH2
where R is CH3xe2x80x94 or H.
In the above formula, the glycolide blocks are the xe2x80x94OCH2xe2x80x94COxe2x80x94 units; addition of a methyl group to the methylene group gives rise to a lactide block; n can be multiples of 2. Vinyl polymerization of the acrylate groups produces an insoluble, crosslinked gel with a polyethylene backbone. The polylactide or polyglycolide segments of the polymer backbone shown above, which are ester groups, are susceptible to slow hydrolytic breakdown, with the result that the crosslinked gel undergoes slow degradation and dissolution. While this approach provides for degradable hydrogels, the structure provides no possibility of covalently attaching proteins or other drugs to the hydrogel for controlled release. Applications of these hydrogels in drug delivery are thus restricted to release of proteins or other drugs physically entrapped within the hydrogel, thus reducing the potential for advantageous manipulation of release kinetics.
Hubbell, Pathak, Sawhney, Desai, and Hill (U.S. Pat. No. 5,410,016, 1995) polymerized:
Protein-NHxe2x80x94PEGxe2x80x94O2Cxe2x80x94CHxe2x95x90CH2
with long wavelength uv radiation to obtain a PEG acrylate polymer with a protein linked to it. The link between the PEG and the protein was not degradable, so the protein could only be hydrolytically released with PEG attached. Since the acrylate polymer is not hydrolytically degradable, the release of the PEG protein derivative is not controllable.
Yang, Mesiano, Venkatasubramanian, Gross, Harris and Russell in J. Am. Chem. Soc. 117, 4843-4850, (1995) described heterobifunctional poly(ethylene glycols) having an acrylate group on one terminus and an activated carboxylic acid on the second terminus. They demonstrated the attachment of this PEG derivative to a protein and incorporation of the resulting PEG protein derivative into an acrylate polymer. However, the PEG backbone there is not degradable and the protein was thus, in effect, permanently bound to the acrylate polymer.
This invention provides heterobifunctional acrylates of poly(alkylene oxides), especially poly(ethylene glycol) (PEG) acrylates having linkages that are hydrolytically degradable and conjugates prepared from these acrylates having target materials such as proteins covalently linked thereto. Hydrogels can also be prepared from these acrylates. The target materials can be released from the hydrogels through controllable hydrolytic degradation of the hydrogels.
In one embodiment of the invention, heterobifunctional PEG is provided which is represented by the formula:
CH2xe2x95x90CZxe2x80x94CO2xe2x80x94PEGxe2x80x94Wxe2x80x94Q
where Z is an alkyl group or hydrogen atom, W is a hydrolytically unstable linkage comprising a hydrolyzable covalent bond, and Q is a reactive moiety capable of reacting with a target to form a covalent linkage thus linking the PEG polymer to the target.
In another embodiment, this invention also provides a heterobifunctional PEG with a hydrolyzable linkage W in the PEG backbone and having an acrylate group at one terminus and a reactive moiety Q at the other terminus. The heterobifunctional PEG is represented by the formula of:
CH2xe2x95x90CZxe2x80x94CO2xe2x80x94PEGxe2x80x94Wxe2x80x94PEGxe2x80x94Q
where Z is an alkyl group or hydrogen atom, W is a hydrolytically unstable linkage comprising a hydrolyzable bond, and Q is a reactive moiety capable of reacting with a moiety on a target such as protein or a drug.
The present invention also encompasses a conjugate having a formula of:
(CH2xe2x95x90CZxe2x80x94CO2xe2x80x94PEGxe2x80x94Wxe2x80x94L)xxe2x80x94T
where Z and W are as described above, T is a target, e.g., a protein or a drug, L is a covalent linkage formed in the reaction between Q and a reactive moiety of T, and x is a number from 1 to 10.
In yet another embodiment of the invention, a conjugate of heterobifunctional PEG and a target is provided having the formula
(CH2xe2x95x90CZxe2x80x94CO2xe2x80x94PEGxe2x80x94Wxe2x80x94PEGxe2x80x94L)xxe2x80x94T
where Z and W are as described above, T is a target, e.g., a protein or a drug, which is linked to the PEG polymer as a result of the reaction between the reactive moiety Q and a moiety on T, L is a covalent linkage formed in the reaction between Q and a reactive group of T, and x is a number of from 1 to 10.
This invention further provides polymers formed by vinyl polymerization of the aforementioned heterobifunctional PEG or conjugates thereof, represented by the formula: CH2xe2x95x90CZxe2x80x94CO2xe2x80x94PEGxe2x80x94Wxe2x80x94Q, (CH2xe2x95x90CZxe2x80x94CO2xe2x80x94PEGxe2x80x94Wxe2x80x94L)xxe2x80x94T, CH2xe2x95x90CZxe2x80x94CO2xe2x80x94PEGxe2x80x94Wxe2x80x94PEGxe2x80x94Q, and (CH2xe2x95x90CZxe2x80x94CO2xe2x80x94PEGxe2x80x94Wxe2x80x94PEGxe2x80x94L)xxe2x80x94T. The weak chemical linkages in the thus formed polymers provide for hydrolytic breakdown and concomitant release of bound target molecules. For example, polymerization of the above-mentioned conjugate:
(CH2xe2x95x90CZxe2x80x94CO2xe2x80x94PEGxe2x80x94Wxe2x80x94PEGxe2x80x94L)xxe2x80x94T
yields a water-soluble acrylate polymer which upon hydrolytic degradation liberates a smaller PEG fragment bearing a target such as a protein or a drug.
In another embodiment of the invention, hydrogels are formed by copolymerizing a heterobifunctional PEG conjugate of this invention with a PEG molecule having two or more acrylate groups (xe2x80x9cPEG multiacrylatexe2x80x9d). Exemplary examples of such PEG multiacrylate can be:
CH2xe2x95x90CHCO2xe2x80x94PEGxe2x80x94O2CCHxe2x95x90CH2
or
xe2x80x83CH2xe2x95x90CHCO2xe2x80x94PEGxe2x80x94Oxe2x80x94CH2CO2CH(CH3)CH2CONHxe2x80x94PEGO2CCHxe2x95x90CH2
The hydrogel of the present invention is a cross-linked network in which protein or other target molecules are covalently bound to a degradable matrix. Because of the hydrolytically unstable linkages W in the hydrogels, the target molecules such as drug or protein molecules can be released as a result of the breakdown of the unstable linkages.
In the heterobifunctional PEG, polymers, and hydrogels of the present invention, the hydrolytic breakdown of the hydrolytically unstable linkages W can be controlled in part by varying W, in particular the number of methylene group proximate to the hydrolyzable bond in W. Specifically, as the number of methylene group increases, the hydrolysis rate of the hydrolyzable bond of W decreases.
Further, in the hydrogel of the present invention, the release rate of the target from the hydrogel can also be controlled by varying the number x in the above formula of the PEG conjugate, i.e., the number of the PEG acrylate linked to the target. The release rate of the target from the hydrogel is decreased when the number of PEG acrylate linked to the target is increased, and vice versa.
Thus, the present invention provides heterobifunctional PEG and hydrogels formed therefrom having target molecules covalently linked to the hyrogels. In contrast to the PEG hydrogels known heretofore in the art, the target molecules can be released in a controlled fashion from the PEG hydrogels of the present invention. Further, since the release rate of the target can be determined by both the number of the attached PEG and the structure of the hydrolytically unstable linkage in the attached PEG, more precise control of the release kinetics is made possible. Therefore, suitable drug carriers for drug delivery which meet different drug release requirements can be made in accordance with the present invention.
The foregoing and other advantages and features of the invention, and the manner in which the same are accomplished, will become more readily apparent upon consideration of the following detailed description of the invention taken in conjunction with the accompanying examples, which illustrate preferred and exemplary embodiments.